A novel GUCY2D mutation in a Chinese family with dominant cone dystrophy.

PURPOSE
To describe the clinical and genetic findings in a Chinese family with autosomal dominant cone dystrophy (adCOD).


METHODS
One family was examined clinically, and genomic DNA was extracted from venous blood of all participants. Genotyping and haplotyping analysis was performed on the known genetic loci for adCOD and autosomal dominant cone-rod dystrophies (adCORD) with a panel of polymorphic markers in this family. All coding exons of the AIPL1, PTTPNM3, and GUCY2D gene were directly sequenced. Allele-specific PCR was used to validate a substitution in all available family members and 100 normal controls. Bioinformatics analysis was done using the Garnier-Osguthorpe-Robson method to predict the effect of the variants detected on the secondary structure of the GUCY2D protein.


RESULTS
Clinical examination and pedigree analysis revealed a three-generation family with four members diagnosed with adCOD. Through genotyping, the disease-causing genes were mapped to chromosomes 17p13.1-2 (AIPL1, PITPNM3, and GUCY2D gene). A novel A->G transition at position 2545 (p.T849A) of the cDNA sequence was identified in the GUCY2D gene. No mutation was detected in the AIPL1 and PITPNM3 genes. This missense mutation co-segregated with the disease phenotype of the family but was not found in the 100 normal controls.


CONCLUSIONS
A novel missense mutation of the GUCY2D gene was identified in this study. Our results further confirm that the dimerization zone of RetGC-1 is the mutational hot region for COD and CORD.

. Family structure, proband fundus appearance, DNA sequence chromatograms, and co-segregation analysis of the p.T849A mutation with the disease phenotype in a Chinese family with cone dystrophy. A: The pedigree and haplotype analysis of the family with cone dystrophy showed segregation with three microsatellite markers on chromosome 17 listed in rising order from the telomere end. Squares indicate males; circles indicate females; slashed symbols indicate deceased; solid symbols indicate affected; open symbols indicate unaffected; M indicates mutant; and + indicates wild-type. B: Fundus appearance of the proband shows the subtle mottling of the RPE in the macula. C: Heterozygote sequence (sense strand) shows an A/G transition in codon 849 that changed threonine to alanine. D: Allele-specific PCR analysis presents the amplified products of the mutation allele (184 bp) co-segregated with patients in this family. The fragments (325 bp), which are the parts of exon3 of the MYOC gene, were used as the internal control in the allele-specific PCR analysis. E: The sequence alignment portion of the dimerization domain spanning the p.T849 of the GUCD2Y of the human with other species.
In this study, we investigated a Chinese family with cone dystrophy. After linkage analysis, we mapped the diseasecausing gene to regions 17p13.1-17p13.2 where the GUCY2D, AIPL1, and PITPNM3 genes are located and found a novel missense mutation of the GUCY2D gene.  examinations, which included bilateral best-corrected visual acuity using E decimal charts, detailed examination of the anterior segment by slit-lamp biomicroscopy, fundus examination with dilated pupils, and a color discrimination test using pseudoisochromatic plates. The proband underwent visual field testing, an electroretinogram, and optical coherence tomography examination.

Patients
Genotyping and haplotyping analysis: Genotyping was performed with 24 microsatellite markers from the autosomes for the known adCOD and adCORD loci in this family (Appendix 1). The fine mapping primer sequences were obtained from the Human Genome Database. Pedigree and haplotype maps were constructed using Cyrillic v. 2.0 software.
Mutation screening of the GUCY2D, AIPL1, and PITPNM3 genes: Mutation screening was performed in the family using direct DNA sequence analysis. All coding regions of the GUCY2D, AIPL1, and PITPNM3 genes were amplified by PCR from the genomic DNA. Primers for all coding exons and exon-intron boundaries of the three genes (18 exons for the GUCY2D, 5 exons for the AIPL1, and 20 exons for the PITPNM3) were designed by the Primer3 program (Appendix 2). For direct sequencing, the PCR products were purified (Shenneng Bocai PCR purification kit; Shenneng, Shanghai, China). The purified PCR products were sequenced using an automatic fluorescence DNA sequencer (ABI, Prism 373A; Perkin Elmer, Foster City, CA) according to the manufacturer's instructions. All PCR products were sequenced in both forward and reverse directions, and the nucleotide sequences were compared with the published DNA sequences of the GUCY2D, AIPL1, and PITPNM3 genes (GenBank accession number NM_000180, NM_014336, and NM_031220, respectively) using DNAssit version 1.0. For the three genes, cDNA numbering +1 corresponded to A in the ATG translation initiation codon of GUCY2D, AIPL1, and PITPNM3, respectively.
Allele-specific PCR analysis: To confirm the variation found in the sequencing, allele-specific PCR analysis (AS-PCR) was performed in the available family members and in 100 normal controls [9]. An allele-specific forward primer in Exon 13 of the GUCY2D gene was designed: 5′-GGA GCT GGA AAA GCA GAA GG-3′, where C is the mutation-specific nucleotide. The AS-PCR fragment was amplified with the forward allele specific primer and the normal reverse primer of the exon13 of the GUCY2D gene.
Bioinformatics analysis: Garnier-Osguthorpe-Robson software was used to predict the effect of the mutation on the secondary structure of GUCY2D (Biotools) [10]. This method infers the secondary structure of a sequence by calculating the probability for each of the four structure classes (helix, sheet, turn, and loop) based on the central residue and its neighbors from the calculated matrices [10]. The PolyPhen2 (Polymorphism Phenotyping 2) program was used to predict the potential functional impact of an amino acid change [11].

Clinical findings:
We identified a three-generation family consisting of four patients diagnosed with cone dystrophy ( Figure 1A). All patients had experienced bilateral visual acuity impairment and marked photophobia in their early childhood. No patients, including the two patients aged over 50 years, had peripheral field loss or a nyctalopia complaint. Slit-lamp examination showed the anterior segments were normal with the exception of mild cataracts in both eyes of one patient (II-1). Fundus examinations revealed subtle RPE granular abnormalities in the macular area and normal appearance of the peripheral retina. A pseudoisochromatic plates test showed red-green color weakness. The proband was examined in 2008 at age 24 years and again in 2011 at age 27 years. His best-corrected visual acuity was 0.5 in both eyes, and there was no significant deterioration during the three following years. Fundus examination showed almost  normal fundus appearance with the exception of subtle mottling of the RPE in the macular area ( Figure 1B). Electroretinographic testing revealed a significant reduction in cone responses and normal rod responses (Figure 2). Optical coherence tomography showed thinning of the retina and loss of the photoreceptor inner segment (IS) and photoreceptor outer segment (OS) in the macular area ( Figure 3). The detailed clinical features are summarized in Table 1.
Genotyping results: This family was genotyped with 24 polymorphic markers around known adCOD and adCORD loci. The mapping results excluded the other known adCOD and adCORD loci with the exception of a locus, 17p13.1-2, where the GUCY2D, AIPL1, and PITPNM3 genes are found.
Mutation analysis: Sequencing of the three genes (GUCY2D, AIPL1, and PITPNM3) revealed one novel heterozygous mutation c.2545 A> G (p.T849A) in the GUCY2D gene.
Using AS-PCR analysis, this mutation co-segregated with the adCOD phenotype in this family and was not detected in the unaffected members or 100 normal controls ( Figure 1C,D).
In addition to the pathogenic mutation detected in the GUCY2D gene, six nonpathogenic sequence variants were also identified in this study. Table 2 summarizes these variants based on their nature.
Bioinformatics analysis: Using the Garnier-Osguthorpe-Robson method, the results of the secondary structure prediction suggested that the mutant GUCY2D 849A replaced one helix, "H," with one β sheet, "E," at position 852. Through PolyPhen-2 program analysis, p.T849A was predicted to be potentially damaging.

DISCUSSION
In this study, we identified one novel missense mutation, p.T849A, in the GUCY2D gene in a small family with adCOD. The mutation co-segregated with the disease phenotype but was absent in the unaffected family members and 100 normal controls.
In the clinical phenotype of the affected members of the family with the mutation p.T849A, the visual acuity of the proband was 0.5 with almost normal fundus. The male subject's electroretinograms demonstrated reduced cone function and nearly normal rod function. Two elder patients (over 50 years old) had preserved peripheral visual fields and no complaints of night blindness. These findings are similar to the previous descriptions of the phenotypes associated with the mutations p.R838C and p.R838H [2][3][4][5]. Since the mutation p.R838C has been identified, the detailed clinical phenotypes with similar or different mutations have been reported by several studies [2][3][4][5]14,16,17,21,22]. Usually, patients with the mutations p.R838C and p.R838H have relatively similar clinical features of COD, which include the marked dysfunction of the cones from a young age while rod dysfunction appears later or does not present until a later stage [2][3][4][5]15,17,18,22,23].
In conclusion, we identified a novel mutation, p.T849A, in a Chinese family with COD. Our results further suggest that the dimerization zone of RetGC-1 is the mutational hot region for COD or CORD.

APPENDIX 1.
Markers used in known autosomal dominant cone dystrophy and autosomal dominant rod dystrophy genotyping. To access the data, click or select the words "Appendix 1."

APPENDIX 2.
Primer information for the AIPL1, PITPNM2, and GUCY2D gene sequence. To access the data, click or select the words "Appendix 2."